Systemic and regional heparinization, to modulate blood coagulation, has played a central role in modern medicine and surgery. However, severe complications can occur due to heparinization. Although many different approaches have been attempted to address this issue, heparin neutralization in blood remains a serious problem. We have been studying and developing a heparin neutralization system (HNS) based on heparinase (E.C.4.2.2.7; an enzyme that specifically depolymerizes heparin). During the past grant period, significant progress has been made in all of the areas proposed in the previous grant application. Specifically we have:1) characterized heparinase at the molecular level, obtained internal peptide sequences, cloned heparinase using Polymerase Chain Reaction, and expressed recombinant heparinase I as a soluble protein in E. coli, 2) designed improved bioreactors and established principles for minimizing blood damage, 3) developed and validated comprehensive in vivo kinetic models to predict reactor performance, and 4) conducted in vivo studies to assess the safety and efficacy of the proposed approach. We believe that there are compelling reasons to address two critical research goals on the heparinase filter. The first area is in improving heparinase stability and capacity in the reactor; the second is in improving the reactor configuration in order to increase the blood flow rate and thus enhance its effectiveness. Our proposed studies involve both in vitro and in vivo studies in these important areas, followed by pre- clinical trials in animal models.